Stimulatory Effects of Extracellular Vesicles Derived from Leuconostoc holzapfelii That Exists in Human Scalp on Hair Growth in Human Follicle Dermal Papilla Cells

Human hair follicle dermal papilla cells (HFDPCs) located in hair follicles (HFs) play a pivotal role in hair follicle morphogenesis, hair cycling, and hair growth. Over the past few decades, probiotic bacteria (PB) have been reported to have beneficial effects such as improved skin health, anti-obesity, and immuno-modulation for conditions including atopic dermatitis and inflammatory bowel disease (IBD). PB can secrete 50~150 nm sized extracellular vesicles (EVs) containing microbial DNA, miRNA, proteins, lipids, and cell wall components. These EVs can regulate communication between bacteria or between bacteria and their host. Although numerous biological effects of PB-EVs have been reported, the physiological roles of Leuconostoc holzapfelii (hs-Lh), which is isolated from human scalp tissue, and the extracellular vesicles derived from them (hs-LhEVs) are largely unknown. Herein, we investigated the effects of hs-LhEVs on hair growth in HFDPCs. We show that hs-LhEVs increase cell proliferation, migration, and regulate the cell cycle. Furthermore, hs-LhEVs were found to modulate the mRNA expression of hair-growth-related genes in vitro. These data demonstrate that hs-LhEVs can reduce apoptosis by modulating the cell cycle and promote hair growth by regulation via the Wnt/β-catenin signal transduction pathway.     

Clinical stage drugs targeting inhibitor of apoptosis proteins purge episomal Hepatitis B viral genome in preclinical models

A major unmet clinical need is a therapeutic capable of removing hepatitis B virus (HBV) genome from the liver of infected individuals to reduce their risk of developing liver cancer. A strategy to deliver such a therapy could utilize the ability to target and promote apoptosis of infected hepatocytes. Presently there is no clinically relevant strategy that has been shown to effectively remove persistent episomal covalently closed circular HBV DNA (cccDNA) from the nucleus of hepatocytes. We used linearized single genome length HBV DNA of various genotypes to establish a cccDNA-like reservoir in immunocompetent mice and showed that clinical-stage orally administered drugs that antagonize the function of cellular inhibitor of apoptosis proteins can eliminate HBV replication and episomal HBV genome in the liver. Primary human liver organoid models were used to confirm the clinical relevance of these results. This study underscores a clinically tenable strategy for the potential elimination of chronic HBV reservoirs in patients.Subject terms: Target validation, Hepatitis B, Preclinical research, Translational research     

Mutational effects on thermostable superoxide dismutase from Aquifex pyrophilus: understanding the molecular basis of protein thermostability

We designed two mutants of superoxide dismutase (SOD), one is thermostable and the other is thermolabile, which provide valuable insight to identify amino acid residues essential for the thermostability of the SOD from Aquifex pyrophilus (ApSOD). The mutant K12A, in which Lys12 was replaced by Ala, had increased thermostability compared to that of the wild type. The T(1/2) value of K12A was 210 min and that of the wild type was 175 min at 95 degrees C. However, the thermostability of the mutant E41A, which has a T(1/2) value of 25 min at 95 degrees C, was significantly decreased compared to the wild type of ApSOD. To explain the enhanced thermostability of K12A and thermolabile E41A on the structural basis, the crystal structures of the two SOD mutants have been determined. The results have clearly shown the general significance of hydrogen bonds and ion-pair network in the thermostability of proteins.     

Conserved cysteine and tryptophan residues of the endothelin-converting enzyme-1 CXAW motif are critical for protein maturation and enzyme activity

The neprilysin (NEP)/endothelin-converting enzyme (ECE) family of metalloproteases contains a highly conserved carboxyl-terminal tetrapeptide sequence, CXAW, where "C" is cysteine, "X" is a polar amino acid, "A" is an aliphatic residue, and "W" is tryptophan. Although this sequence strongly resembles a prenylation motif, human ECE-1 did not appear to be prenylated when labeled in vivo using various isoprenoid precursors in cell lines expressing ECE-1. We used site-directed mutagenesis to investigate the role of the CXAW motif and determined that the conserved cysteine residue of the CXAW motif in ECE-1, Cys(755), is critical for proper folding of the enzyme, its export from the endoplasmic reticulum, and its maturation in the secretory pathway. In addition, site-directed mutagenesis revealed that the conserved tryptophan residue of the sequence CEVW appears to be important for endoplasmic reticulum export and is essential for enzyme activity. Deletion of Trp(758) or substitution with alanine greatly slowed maturation of the enzyme, and resulted in more than a 90% loss of enzyme activity relative to the wild type. Conservative substitution of the tryptophan with phenylalanine did not reduce activity, whereas replacement with tyrosine, methionine, or leucine reduced enzyme activity by 50%, 75%, and 85%, respectively. Together, these data indicate that the conserved CEVW sequence does not serve as a prenylation signal and that both the conserved cysteine and tryptophan residues are necessary for proper folding and maturation of the enzyme. Furthermore, the conserved tryptophan appears to be critical for enzyme activity.     

Nudf, a fungal homolog of the human LIS1 protein, functions as a dimer in vivo

The NUDF protein is required for nuclear migration through the mycelium of the filamentous fungus Aspergillus nidulans. It is of particular interest, because it closely resembles a human protein, LIS1, that is required for development of the cerebral cortex. Both are approximately 50-kDa proteins with a short N-terminal predicted coiled coil and seven WD-40 domains in the C-terminal half of the molecule. They also interact with homologous proteins, suggesting that they may have similar biochemical functions. Here we describe the purification to homogeneity of NUDF protein in a single step from a cell-free extract of A. nidulans. We demonstrate that NUDF is a homodimer, that its dimerization occurs via the N-terminal coiled coil region of the molecule, and that it must be a dimer to support the growth of A. nidulans.     

Epidermal growth factor (EGF) receptor-dependent ERK activation by G protein-coupled receptors: a co-culture system for identifying intermediates upstream and downstream of heparin-binding EGF shedding

"Transactivation" of epidermal growth factor receptors (EGFRs) in response to activation of many G protein-coupled receptors (GPCRs) involves autocrine/paracrine shedding of heparin-binding EGF (HB-EGF). HB-EGF shedding involves proteolytic cleavage of a membrane-anchored precursor by incompletely characterized matrix metalloproteases. In COS-7 cells, alpha(2A)-adrenergic receptors (ARs) stimulate ERK phosphorylation via two distinct pathways, a transactivation pathway that involves the release of HB-EGF and the EGFR and an alternate pathway that is independent of both HB-EGF and the EGFR. We have developed a mixed culture system to study the mechanism of GPCR-mediated HB-EGF shedding in COS-7 cells. In this system, alpha(2A)AR expressing "donor" cells are co-cultured with "acceptor" cells lacking the alpha(2A)AR. Each population expresses a uniquely epitope-tagged ERK2 protein, allowing the selective measurement of ERK activation in the donor and acceptor cells. Stimulation with the alpha(2)AR selective agonist UK14304 rapidly increases ERK2 phosphorylation in both the donor and the acceptor cells. The acceptor cell response is sensitive to inhibitors of both the EGFR and HB-EGF, indicating that it results from the release of HB-EGF from the alpha(2A)AR-expressing donor cells. Experiments with various chemical inhibitors and dominant inhibitory mutants demonstrate that EGFR-dependent activation of the ERK cascade after alpha(2A)AR stimulation requires Gbetagamma subunits upstream and dynamin-dependent endocytosis downstream of HB-EGF shedding and EGFR activation, whereas Src kinase activity is required both for the release of HB-EGF and for HB-EGF-mediated ERK2 phosphorylation.